Injectable solutions or suspensions of antigens in optically clear colloidal solutions



United States Patent 3,384,544 INJECTABLE SQLUTIONS 0R SUSPENSIONS OF ANTIGENS EN QPTICALLY CLEAR COL- LOIDAL SOLUTIONS Clive A. Walton, Harrow, and Clarence L. 5. Coles and Ernst L. Neustadter, Ruislip, England, assiguors to Glaxo Laboratories Limited, Greeniord, Middlesex, England, a British company No Drawing. Filed June 1, 1967, Ser. No. 642,727 Claims priority, application Great Britain, June 8, 1966,

17 Claims. (Cl. 167-78) ABSTRACT OF THE DISCLOS There are disclosed injectable compositions for veterinary or human medicine in which an antigen is dissolved or suspended in a particular type of vehicle. This vehicle comprises a physiologically acceptable lipophilic dispersion medium, water and at least one physiologically acceptable nonionic surface active substance. It exists in the form of an optically clear colloidal solution in which the aggregates of water and surface active substance are of a size Within the range of 50-800 A. and the ratio of water to surface active substance is within the range of 1:1 to 1:10. The water content of the composition is within the range of 0.522.5% by weight.

This invention concerns a novel type of injectable composition of use in medicine and incorporating one or more high-molecular weight physiologically active substances.

In investigating the adjuvant properties of oily substances in injectable vehicles, it has previously only been proposed to use anhydrous oil suspensions or emulsions of the simple or multiple Water-in-oil or oil-in-water type. Adjuvants of this type, however are often unstable on storage or too thick easily to be injected. Furthermore, an emulsion or a suspension of a hydrophilic substance in oil, being opaque, is a somewhat inelegant product and is difficult to measure accurately in a syringe.

The present invention is particularly concerned with the problem of formulating high molecular weight, hydrophilic, oil-insoluble physiologically active substances such as bacterial toxoids and other antigens in an oily medium without losing the desired physiological activity and without encountering the above-described difficulties associated with the use of emulsions.

We have now found that optically clear solutions of non-ionic amphiphilic surface active material and Water in a physiologically acceptable lipophilic dispersion medium (in contrast to the previously proposed cloudy or opaque emulsions) have the remarkable property of holding in solution a wide variety of high molecular weight hydrophilic substances such as antigens, polysaccharides, nucleic acids, etc., and of releasing such physiologically active substances on injection. Such media are also particularly advantageous as delayed release vehicles for insoluble antigens such as living or dead micro-organisms.

According to the present invention we provide injectable compositions consisting of or containing water in solution in a physiologically acceptable lipophilic dispersion medium, which is liquid at body temperature, by means of one or more non-ionic, amphiphilic, physiologically acceptable surface active substances, said water having dissolved or suspended therein one or more physiologically-active micro-organisms and/or physiologically active high molecular weight hydrophilic oil-insoluble materials.

The term solution is used herein to indicate a system in which the water is held by the surface-active material in a continuous lipophilic dispersion medium and that the resulting systems are virtually optically clear when viewed by transmitted light, as distinct from emulsions or suspensions which are cloudy or opaque, unless additional components such as thickening agents are added which themselves lend opacity to the solutions.

Optically clear solutions of the kind utilised in the present invention include all colloidal solutions wherein the aggregates of water and surface-active materials are of a diameter below about 800 A. and thus no longer cause appreciable opacity to visible light. The size range of the aggregates is thus approximately 50-800 A.; above about A. within this range the solutions are sometimes termed microemulsions. This is in contradistinction to normal or macroemulsions wherein the droplet size is of the order of 2,000 A. The conditions required for formation of solutions (including the so-called microemulsions) are such that the resulting aggregates are thermodynamically stable, in contrast to macroemulsions which are necessarily thermodynamically unstable, even though the equilibrium conditions of phase separation may be greatly delayed (L. I. Osipow, J. Soc. Cosmetic Chem. 1963 14, 277-288; L. M. Prince, J. Colloid and Interface Science, 23, -173).

The physiologically active material may itself be solubilised in the above sense to give a clear solution or may remain in suspension; micro-organisms of course will always he in suspension. Even suspensions of the above kind may sometimes be virtually optically clear in transmitted light if the refractive index of the organisms is close to that of the mediuml The new compositions, being in general optically clear solutions, are not only more elegant in appearance than emulsions or suspensions but have manufacturing advantages. They are far easier to sterilise because filtration methods may be employed and the preparations are, in general, more stable to transport vibration and temperature fluctuation on storage. They may be readily produced by simply mixing the components without energetic homogenisation which could in somecases be detrimental to the physiologically active substances. These factors are of considerable economic value. In addition, the solutions can be prepared to have viscosity levels permitting easy handling and injection.

Surprisingly, considerable quantities, for example of the order of 20% W./v. or more, of aqueous solutions or suspensions of physiologically active materials may be held in the compositions according to this invention and substantially non-viscous compositions, suitable for injection through a narrow bore needle, may be prepared quite simply, without the need for expensive equipment for particle size reduction and control in the case of suspensions, or homogenising machinery in the case of emulsions, which processes are diflicult to control. However, thicker compositions may be obtained if required, by varying the nature of the wetting agent and lipophilic dispersion medium.

The compositions according to the invention may thus include one or more physiologically active high molecular weight susbtances. These substances will preferably be non-dialysable, their molecular weight being preferably above 1,000, and they include, for example, antigenic substances, e.g., bacterial toxoids, etc., proteins, polysaccharides, nucleic acids, etc. The microorganisms which may be present can be, for example, living or dead bacteria or viruses. Thus, for example, in the veterinary field,

Cl. welchii types B, C and D, Cl. oedematiens, Cl. chauvoez', Cl. return and Cl. septz'cum.

The amount of hydrophilic oil-insoluble substances that can be dissolved in the compositions according to this invention will depend in each case on the components that are present, especially the surface-active components.

Frequently the use of more than one surface-active agent in a mixture of the appropriate H.L.B. will enable more hydrophil-ic material to be dissolved than would be the case using a single surface-active agent of the ame H.L.B. value and it is particularly advantageous to add further to a suitable mixture of surface-active agents a surface liquidiser. Such surface liquidisers will usually be amphiphilic substances of shorter chain length than the principal surfactant and may be thought of as exerting a lubricating effect by becoming interposed between the longer amphiphilic molecules. Mixtures according to this invention containing such surface liquidisers are usually considered to contain larger aggregates in the range 100-800 A.

Such amphiphilic surface liquidisers include physiologically acceptable fatty acid aldehydes, ketones and, in particular, amphiphilic alcohols, for example mono, dior polyhydric alcohols having 3-10 carbon atoms, e.g., n-decanol, 2-ethyl-hexane-l,3-di0l, 4-methyl-cyclohexanol, etc.

The weight ratio of added surface liquidiser to total amphiphilic material advantageously employed to obtain transparent stable solutions is strongly dependent on the temperature range over which clarity is required. The amount is also dependent on the nature of the oil and other amphiphilic surface-active agents employed. In our preferred mixtures We have found the percentage by weight of surface liquidiser based on the total amphiphilic material for clarity at body temperature advantageously to be 40%, preferably from 025%. It may be noted that the minimum quantity of amphiphilic surface-active material required to produce a clear solution may often be lower when a surface liquidiser is present.

As indicated above, the new vehicle used in the compositions according to the invention is optically clear and is capable of holding considerable quantities of hydrophilic substances in a clear solution in oil. Such solutions may readily be prepared in a thin non-viscous form suitable for injection in contrast to the conventional waterin-oil emulsions which are usually too thick to be easily injected. However, by varying the nature of the wetting agent and the lipophilic dispersion medium, thicker solutions may be obtained.

The lipophilic dispersion medium may, for example, be an oil which is liquid at body temperature. In general, however the lipophilic component is preferably liquid at 35 (3., more preferably at room temperature and below, to facilitate handling of injectable preparations.

Name

1. Arlaeol 20 Arlacel 80- Arlacel A.

Triton X 100: Polyoxyethylene (10) octyl pheno fatty acid esters or mixtures thereof such as tridecyl myristate, n-octyl oleate or vegetable oils such as coconut oil.

When the lipophilic material is an ester or a straight or branched chain aliphatic hydrocarbon, such as paraffin oil or squalane, the surface active material preferably possesses an H.L.B. (hydrophile-lipophile balance) value in the range 7 to 12, advantageously between 8 and 11, the optimal value being between 8.5 and 10. It should be noted that where a mixture of surface active agents is used, it is the H.L.B. value of the mixture which should fall within the above range.

The preferred surface active agents fall in the follow ing four classes:

(1) Fatty acid esters of sugar alcohol anhydrides, for example of sorbitan or mannitan. Fatty acid moieties in such substances include oleate, stearate, laurate residues etc. Soroitan mono-oleate and mannitan mono-oleate are especially useful and mannitan mono-oleate is obtainable in a. specially purified grade widely used in injectable preparations. Commercial products of this class include Arlacel A (mannitan mono-oleate), Arlacel 8O (sorbitan mono-oleate) and Arlacel 2O (sorbitan mono-laurate).

(2) Ethylene oxide condensates of the products of Class 1. Polyoxyethylene sorbitan mono-oleate and monolaurate are particularly useful. Commercial products of this class include Tween (polyoxyethylene (20) sorbitan mono-oleate), Tween 20 (polyoxyethylene (20) sorbitan mono-laurate), Tween 81 (polyoxyethylene (5) sorbitan mono-oleate), Tween 85 (polyoxyethylene (20) sorbitan trioleate), Tween 61 (polyoxyethylene (4) sorbitan mono-stearate) and Tween 65 (polyoxyethylene (20) sorbitan tristearate). The numerical values given in parentheses in the nomenclature for the above products refers to the approximate number of oxyethylene units. The products are, in fact, always mixtures and this figure merely represents the average chain length.

(3) Polyoxyethylene derivatives of alkyl phenols. The alkyl portions of such phenols preferably contain 6 to 10 carbon atoms, e.g., as in octyl or nonyl groups. Products of this type having polyoxyethylene chains of varying lengths are commercially available. Commercial products in this class include Triton X-15 (polyoxyethylene (1)-octyl phenol), Triton X-35 (polyoxyethylene (3)-octyl phenol) and Triton X-lOO (polyoxyethylene (l0)-octyl phenol).

(4) Polyoxyethylene derivatives of fatty alcohols, e.g., lauryl, stearyl alcohol etc. Again, materials of varying chain lengths are obtainable. Brij 30 (polyoxyethylene (4)-lauryl ether), is a useful product in this class.

Naturally the surface active material and the lipophilic material must be compatible with the biologically active component.

The H.L.B. values of a number of surface active materials are given in Table 1 below.

TABLE L-SURFACE, ACTIVE AGENTS, EXAMINED Chemical Constitution sorbitan monolaurato sorbitan mouoo1eate Mannitan monooleate Polyoxyetnylene (20) sorbitan monolaurate Polyoxyethylene (20) sorbitan monooleate Polyoxyothylene (5) sorbitan monooleate. Polyoxyethylene (20) sorbitan trioleate Polyoxyethylene (20) sorbiton monostearate Polyoxyethylene (4) sorbitan m0n0stearate.... Polyoxyethylene (20) sorbitan tristearate Polyoxyethylcne (l) octyl phenoL. Polyoxycthylene (3) octyl phenol H.L.B.

Polyoxyeth ylene (4) lauryl other In order to obtain an optimal H.L.B. value it is often convenient to use a mixture of a predominantly hydrophilic surface active agent with a predominantly lipophilic surface active agent and Table H below shows results obtained with a number of such mixtures and also Tween 81. Weighed quantities of surfactant solutions sealed in ampoules with incremental quantities of Water were mixed and examined for clarity to determine the phobic components using insufficient surface active agent maximum quantities of water solubilised. Other tech to solubilise all the water and then to evaporate off a niques show higher values and those shown are principroportion of the water to leave a solubilised preparation. pally for the purpose of comparing the properties of the Evaporation of the water can be effected, for example, surface active agents. 5 by passing a current of sterile air over the surface of the TABLE II.QUANTITIES (PERCENTAGE WJW.) OF WATER SOLUBILISED AT ROOM TEMPERATURE Composition Concentration of surface active agent (percent w./w.)

Number Surface active agents Ratio H.L.B. Oil phase 7. 5 1 1 1 Liquid paraffin. as 5.5 7.5 Do. 9.6 4.5 4. 46 Do. 10.7 3.4 5.5 Do. 8.3 2.3 3.4 Do. 9.5 2.3 3.4 Do. 10.5 2.3 3.4 Do. 11. 2 s. 52 10. a Do. 11.3 2. 3-5.5 2. 3-8.45 Do. 12.5 1 1-.. 1 5.4-7.4 Do.

8.6 0.56 only.. U.56-1.1. 2.21 3.24 n-octy oleate. 9.6 0.56 only- Nil... "055-11-..- 3.3 Do. 10.7 Nil Nil 221 only 0. 55-3. 25 Do. do 8.6 1.2 only. 1.23-24 0.6-4.6 Squalane. 15 Tween 81 10.0 N11 2.3 4.5 .6 Liquid parafiin.

From these tables it can be seen that surface active agitated emulsion or by evaporation at low temperatures materials based on sorbitan give especially good results under high vacuum. in that they solubilise relatively large quantities of w&- The compositions according to the invention may, if

ter. The experiments indicate that Tween 81 alone, Arlacel 25 desired contain additional components such as bacteriosta- 801Tween 80 (60:40) and Arlacel 20:Tween 80 (60:40) tics, antiseptics etc. or thickening agents for viscosity conare especially effective in a liquid parafiin dispersion metrol.

dium. Brij is another useful surface active material. The solubilised compositions according to the invention It will be noted that the H.L.B. values of these surface can in some cases be modified by heating to relatively active combinations are all about 10. 30 high temperatures, for example above 40 C., whereby As indicated previously, the preferred HLB. values water separates out to give a turbid appearance. It is prestated above are those which apply when the lipophilic ferred, therefore, that the compositions remain clear on medium is a straight or branched chain hydrocarbon and, heating to at least body temperature. It should be borne especially, paraifin oil or squalane. Where other lipophilic in mind that some animals have a relatively high body media are selected, the optimal H.L.B. values will differ temperature, for example the body temperature of sheep although they can readily be ascertained by experiment. is normally around C.

The percentage of water in the compositions may vary The new compositions according to the invention are widely and up to about 22.5% water can be incorporated intended for pharmaceutical and veterinary use. The virin oils such as liquid parafiin while still maintaining a tually clear compositions according to the invention in adclear solution. On the other hand, the association of a large 40 dition to the physical advantages described above have quantity of water with the physiologically active material also shown surprisingly marked adjuvant effects on the may not be necessary and as little as 0.5 water or even properties of the active material. Thus, for example, in less may be present. Where the high molecular weight the case of Cl. welchii, type D formol toxoid, the height component is difficult to obtain in concentrated aqueous of the antibody response was increased in our experiments solution, as is often the case with bacterial toxoids and by a factor of ten and the duration of protection was also particularly where a mixture of several toxoids is reincrease-d. In the administration of veterinary vaccines, quired, it is preferred that the percentage of water should and indeed human vaccines, it is important that the durabe, for example 10 to 15% with Tween 81 as surfactant. tion of protection be as long as possible and if the num- Such percentages can be obtained by using relatively large her of injections necessary to give protection can be miniquantities of surface active agent. mised this is of great benefit in reducing the cost of pro- Where concentrated solutions of the active component tecting large numbers of animals. While we do not wish are available, however, it may be preferred that the perto be bound by theoretical considerations, it is believed centage of water be kept relatively low, for example in that the increased effectiveness of the active material is the range 0.5% to 7%, more preferably between 2.5% and due to delayed release from the lipophilic medium. 6.0%, so that the amount of surface active agent present For the better understanding of the invention the folcan be minimised. The weight ratio of water to surface lowing examples are given by way of illustration only: active agent is preferably in the range 1:1 to 1:10, advantageously 1:4 to 1:7, for example about 1:5. EXAMPLE 1 In the field of human medicine, physiologically active Percent material which may be incorporated into the composition Arlacel 80 (sorbitan mono-016MB) includes tetanus, diphtheria and staphylococcal toxoids as 69 Tween 20 (polyoxyethyelene sorbitan IIIOIIO- well as suspensions of organisms such as B. pertussis, V. laufate) 3- 5 cholerae, i fl virus m Closlridium welchii type D, purified formol toxoid The compositions of the invention may be prepared in of a P y Of 4,500 -f m1 1.45 a number of ways. As indicated above, it is possible for Puremor (extra llght Whlte P a n Oil) to by volthe surface active agent to be dissolved in the oil and for 100 the aqueous material to be added thereto, preferably slow- M thod of preparation ly. It is also possible to mix the aqueous components with the hydrophobic phase and to add surface active material to produce solubilisation. The surface active agent may 70 (1) A solution of 10 grams of Arlacel 80 in sufiicient Puremor to produce 191 ml. was sterilised by passage through a Millipore membrane filter.

also be added first to the aqueous Component and (2) Tween 20 was sterilised by autoclaving at 10 p.s.i. hydrophobic 'P mixed Subsequently therewith one (3) Tween 20 (3.25 ml.) was aseptically measured into further methOdis especially useflllwhefe the Physiological 95.5 ml. of sterile Arlacel 80 solution, and the toxoid ly active material is available only in dilute solution, namesolution added. The mixture was stirred until homogeneous ly to prepare an emulsion of the aqueous and hydroand packed.

7 EXAMPLE 2 Percent Tween 81 polyoxythylene sorbitan monooleate w./v

Clostridium welchii type D, purified formol toxoid solution containing 4,000 Lf/ml. v./v Puremor extra light white parafiin oil, to

by volume Method of preparation (1) 15.0 grams of Tween 81 was dissolved in sufficient Puremor to produce 148 ml. of solution. This solution was sterilised by passage through a sterile Millipore membrane filter.

(2) To 99 ml. of the sterile solution was added 1.25 ml. of toxoid solution. The mixture was stirred until homogeneous and packed.

EXAMPLE 3 Triton X-100 (polyoxyethylene octyl phenol) w./v 9.0 Triton X-lS (polyoxyethylene (1) octyl phenol) w./v 9.0 Clostridium welchii Type D, purified formol toxoid at 4,000 Lf/ml v/v 6.0

Puremor extra light white parafiin oil, to by volume 100 Method of preparation (1) 18.0 g. Triton X- dissolved in Puremor to produce 100 ml., was sterilised by filtration.

(2) Triton X-100 was sterilised by autoclaving at 10 p.s.i.

(3) 9.0 g. Triton X-100 was mixed aseptically with 50 ml. Triton X-l5 solution. The toxoid was added and the product made to 100 ml. with sterile Puremor.

(4) The product was then stirred until homogeneous.

EXAMPLE 4 Formula, as in Example 2.

Method of preparation (1) To a crude emulsion of toxoid solution in sterile Puremor, was added the sterile Tween 81, stirring continually with a mechanical stirrer. The product was stirred until homogeneous.

EXAMPLE 5 Formula as in Example 2.

Method of preparation (1) Toxoid solution was added to and mixed intimately with sterile Tween 81. This mixture was. then diluted with Puremor to volume. The product was stirred until homogeneous.

EXAMPLE 6 Percent Tween 81 W./v 10.0 Clostridium welchii type D toxoid at 3,600 Lf/ml.

v./v 1.39 Thixin R (glyceryl tris-IZ-hydroxystearate) w./v 1.0 Puremor, to by volume..- 100 Method of preparation (1) A w./v. solution of Tween 81 in puremor was sterilised by filtration.

(2) 10 grams of Thixin -R sterilised by exposure to formalin vapour, was dispersed in sterile Puremor to produce 200 grams of suspension. The suspension was warmed to 70 C. to dissolve the Thixin -R. and the mixture stirred vigorously until it had cooled to ambient temperature.

(3) 1.39 ml. toxoid was added to 50 ml. of Tween 81 solution and 20 grams of Thixin gel added. The mixture was stirred until homogeneous, and diluted to 100 ml.

with sterile Puremor. (Note that this vaccine was not clear, the Thixin-R shown in this formula was incorporated as a thickening agent, and does not produce a clear solution. It was incorporated against possible inclusion of cellular antigens together with toxoids, to determine its acceptability in terms of antitoxin response to toxoids.)

EXAMPLE 7 Tween 81 g./l 0.18 Tween (polyoxyethylene (20) sorbitan monooleate) g./l 0.02

Mixture of suitable solutions to contain, for each ml. of

vaccine these quantities:

Puremor, to 1.0 ml.

Method of preparation (1) A mixture of toxoids to the proportions shown were freeze dried, and reconstituted in water for injection to produce 5 ml. of antigen solution per ml. of vaccine.

(2) A solution in Puremor containing 18 grams of Tween 81 and 2 grams of Tween 80 per 75 ml. of solution was sterilised by filtration.

(3) The mixed antigen solution was added to this solution and the product made to 100 ml. with sterile Puremor and stirred until homogeneous.

EXAMPLE 8 Percent Tween 81 w./v 15.0 Brucella abortus strain 45/20, packed cells w./v 5.0 Thixin-R w./v 1.0 Puremor, to by volume 100 Method of preparation (1) The packed cells (which contain approximately 50% water) were dispersed thoroughly in sterile Tween 81.

(2) This dispersion was then dispersed in sterile Puremor 25 g. of 4% Thixin-R gel, prepared as described under Example 6, was added, and the product made to volume with sterile Puremor. The product was then agitated until homogeneous.

EXAMPLE 9 Percent Tween 80 w./v 1.5 Tween 81 w./v 13.5 Clostridium welchii type D forrnol toxoid at 3,000

Lf/ml. v./v 1.66 Squalane, to by volume-.. 100

Method of preparation (1) Tween 80 and Tween 81 were autoclaved at 10 p.s.i. for 30 min. to sterilise.

(2) The Tweens were aseptically dissolved in sterile (filtered) Squalane, and the toxoid solution added. The product mechanically stirred until homogeneous.

9 EXAMPLE 10 Formula: Percent Infectious Bronchitis virus suspension v./v 20 Tween 81 1 w./v 20 Puremor to by volume 100 1 sterilised by filtration.

Method of preparation (using sterile equipment and aseptic technique) (1) 42.5 ml. of a 23.55 percent w./v. solution of Tween 81 in Puremor was measured into a jacketed glass vessel, equipped with a ground glass lid and magnetic stirrer.

(2) 10 ml. of virus suspension was added and stirred to produce a water in oil emulsion.

(3) A glass freeze drying trap containing a Drikold- IMS mixture was inserted in the socket provided in the lid of the vessel.

(4) Vacuum, approximately 28 inches of mercury, was applied to the system, and water at 27 C. circulated through the jacket. Water was condensed from the system, stirring continually, until the product was clear. The product was made to 5 ml. with Puremor.

The moisture content of the product was measured as 11.7 percent w./w.

EXAMPLE 11 Formula, as Example 10.

Method (Using sterile materials and equipment and an aseptic technique) EXAMPLE 12 Tetanus vaccine: Percent Tween 80 W./v 2.0 Tween 81 w./v 18.0 Clostridium tetani purified formol toxoid solution at 150 Lf/ml. v./v 5.0 Puremor, to 100.0

(1) 36 g. of Tween 81 and 4 g. of Tween 80 were dissolved in sufficient Puremor to produce 190 mls. of solution. This solution was sterilised by passage through a sterile Millipore membrane filter.

(2) To 95 mls. of the sterile solution was added mls. of toxoid solution. The mixture was stirred until homogeneous and packed.

EXAMPLE 13 Pertussis vaccine: Percent Tween 81 w./v 20.0 Cell suspension containing 200x B. Pertussis organisms/ml. v./v 5.0 Puremor, to 100.0

(1) 40 g. of Tween 81 was dissolved in sufiicient Puremor to produce 190 mls. of solution. This solution was sterilised by filtration.

(2) To 95 mls. of the sterile solution was added 5 mls. of cell suspension. The vaccine was stirred until homogeneous and packed.

EXAMPLE 14 Tween 60 (polyoxyethylene sorbitan monostearate) g 10 Arlacel 8O (sorbitan monoleate) g 10 2-ethy1-1,3-hexanediol g 4.8 Mixture of suitable solutions to contain, for each ml.

of vaccine (11.6 ml.), these quantities:

Clostridium oedematiens type B formol toxoid Lf 5 Puremor g 47 Method of preparation (1) A solution of 10 g. Tween 60, 10 g. Arlacel and 4.8 g. 2-ethyl-1,3-hexanediol in 47 g. Puremor was sterilised by filtration.

(2) The above solution was warmed to 40 and the mixed antigen added with stirring. The product was stirred until clear and allowed to cool.

It was clear over a range 1045 C. (The final volume was 93 ml.)

EXAMPLE l5 Tween 60 g 12 Arlacel 80 g 12 Mixture of suitable solutions to contain, for each ml.

of vaccine these quantities:

Clostridium welchii type B purified toxoid (formol) Lf Clostridium welchii type C purified toxoid (formol) Lf 50 Clostridium welchii type D purified toxoid (formol) Lf 50 Clostridium tetani purified formol toxoid Lf 7.5 Clostridium septicum purified formol toxoid Lf -7.5 Clostridium oedematiens type B formol toxoid LL- 5 Clostridium chauvoei formol culture 3 10 organisms. 2-ethyl-1,3-hexanediol g 8.5 Coconut oil, to mls 100 A mixture of toxoids to the proportions shown were freeze dried and reconstituted in water for injection to produce 6.7 mls. of antigen solution per 100 ml. vaccine.

Method of preparation (1) A solution of 10 g. Tween 60, 10 g. Arlacel 80 and 4.8 g. 2-ethyl-1,3-hexanediol in 47 g. Puremor was sterilised by filtration.

(2) The above solution was warmed to 40 and the mixed antigen added with stirring. The product was stirred until clear and allowed to cool.

We claim:

1. An injecta'ble composition, comprising a parenterally effective quantity of at least one physiologically active antigenic substance dissolved in a liquid vehicle consisting essentially of an optically clear colloidal solution containing a physiologically acceptable lipophilic dispersion medium liquid at 35 C. selected from the group consisting of aliphatic hydrocarbons and long chain fatty acid esters, water and :at least one physiologically acceptable non-ionic amphiphilic surface active substance, the aggregates of Water and surface active substance having a size range between 50-800 A., the ratio of water to surface active substance being within the range of 1:1 to 1:10, and said composition containing from 0.5-22.5% by weight of water.

2. A composition -as claimed in claim 1 which contains microorganisms in suspension as an antigenic substance.

3. A composition .as claimed in claim 2 in which said microorganisms are B. aborms or B. pertussis.

4. A composition as claimed in claim 1 in which said antigenic substance is a bacterial toxoid.

5. A composition as claimed in claim 4 in which said toxoid is at least one member selected from the group consisting of diphtheria .toxoid, staphylococcal toxoid, a toxoid from Clostridium welchii types B, C or D, a toxoid from Closrridz'um oedematiens, a toxoid from Clostridium septicum, and a toxoid from Clostridium tetani.

6. A composition as claimed in claim '1 in which said lipophilic dispersion medium is a hydrocarbon selected from the group consisting of aliphatic and cycloaliphatic hydrocarbons.

7. A composition as claimed in claim 5 in which the hydrocarbon is a member selected from the group consisting of purified paraflin oil and squalane.

8. A composition as claimed in claim 1 in which said lipophi'lic dispersion medium is at least one long chain ester.

9. A composition as claimed in claim 7 in which said ester is selected from the group consisting of tridecyl myrist-ate, n-octyl oleate and .a vegetable oil.

10. A composition as claimed in claim 8 in which said ester is coconut oil.

11. A composition as claimed in claim 1 in which said surface active substance is .at least one member selected from the group consisting of a fatty acid ester of a sugar alcohol anhydride, an ethylene oxide condensate of said ester, a polyoxyethylene derivative of an a-lkyl phenol and a polyoxyethylene derivative of a fatty alcohol.

12. A composition as claimed in claim 1 in which said surface active substance has an H.L.B. value between 7 .and 12.

13. A composition as claimed in claim 10 in which said surface active substance is a mixture of about 40 parts by weight of polyoxyethylene (20) sorbitan monooleate and about 60 parts by weight of a member sclected from .the group consisting of so'rbitan mono-oleate and sorbitan monolaurate.

14. A composition .as claimed in claim 10 in which said surface active substance is polyoxyethylene (5) sorbitan mono-oleate.

15. A composition as claimed in claim 1 in which said non-ionic amphiphilic surface active substance is at least one surface active substance selected from the group consisting of .a fatty acid ester of a sugar alcohol anhydr-ide, an ethylene oxide condensate of said ester, a polyoxyethylene derivative of an alkyl phenol and a polyoxyethylene derivative of a fatty alcohol, the composition containing up to 40% by weight based on the total amphiphilio material of .an .amphiphilic substance of a shorter chain length than said surfiace active substance.

16. A composition as claimed in claim 15 in which said amphiphilic substance of shorter chain length is an alcohol selected from the group consisting of monohydric, dihydric and polyhydric alcohols, said alcohol having 3-110 carbon atoms.

17. A composition as claimed in claim 1 in which the water content is between 2.5 and 6.0% by weight.

References Cited UNITED STATES PATENTS 2,966,443 12/1960 Cox 167-78 3,096,249 7/1963 Prigal 167-82 3,099,601 7/1963 Davis et al. 167-78 3,149,036 9/ 1964 Woodhour et al. 167-78 Re. 25,721 1/ 1965 Howell et al. 167-78 "3,185,625 5/1965 Brown 167-82 3,240,670 3/1966 Feinberg 167-58 LEWIS GOT-TS, Primary Examiner.

S. K. ROSE, Assistant Examiner.

gg UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,384,544 Dated May 21, 1968 Inventor-(s) Clive A. Walton, Clarence L-J. Coles and Ernst L. Neustadter It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 11, claim 9, line 1,

"7" should read "8".

Column 11, claim 13, line 1,

"10" should read "11" Column 12, claim 14, line 1,

" 10" should read "ll" SIGNED Am! SEALED UEC 2 1959 (SEAL) mum 1:. sum, .m. AflfillillgOificer flomisaioner of Patents 

